The role of the transsulfuration pathway in spermatogenesis of vitamin D deficient mice

Vitamin D deficiency is a global health problem and has been linked to defective spermatogenesis and male infertility. In this study, we aimed to investigate the main enzymes involved in the transsulfuration pathway of 1-carbon metabolism, and spermatogenesis function. Therefore, sixteen male C57 mice were addressed to a control (standard diet) or vitamin D deficient (VDD) diet for 14 weeks. The results show that compared to the standard diet, VDD increased final body weight and reduced sperm quality, caused damage to the testicular structure, and decreased the serum levels of testosterone. In addition, serum concentrations of homocysteine, vitamin B12, and sperm oxidative stress markers increased. In testicular tissues, the CBS and CSE protein levels were down-regulated whereas HO-1 was up-regulated at both mRNA and protein expression levels. Within a mice deprivation model, VDD deeply suppressed testosterone and impaired spermatogenesis with oxidative stress-mediated mechanisms. The effects of the deprivation appeared to be at least in part independent of genomic and receptor-mediated vitamin D actions and suggest a specific impairment of the alternative transsulfuration pathway.


Effects of vitamin D deficiency on sperm chromatin status
The harmful effect of VDD on sperm chromatin and DNA status is depicted in Fig. 3a-c.In the VDD group the mean percentage of sperm chromatin protamine deficiency (58.75 ± 6.63 vs 12.00 ± 6.04, P < 0.001), and the percentage of sperm with excessive residual histones (88.62 ± 6.76 vs 26.0 ± 8.12, P < 0.001) were remarkably increased compared to the control group.In addition, the sperm DNA damage increased significantly in mice with VDD (78.87 ± 11.93) compared to the controls (12.80 ± 8.32, P < 0.001).www.nature.com/scientificreports/

Effects of vitamin D deficiency on testicular cells apoptosis
In this study, the TUNEL assay was used to evaluate apoptotic markers in testicular tissue sections.The results depicted in Fig. 3d-f indicate that the VDD group exhibited a higher number of apoptotic cells (42.80 ± 8.92 vs 11.80 ± 6.01, P = 0.0002) and intensity (943.7 ± 137.9 vs 268.7 ± 56.98, P < 0.0001) compared to the control group, indicating significant apoptotic damage.

Effects of vitamin D deficiency on serum metabolites
Our diet was effective in inducing a shortage of vitamin D (14.66 ± 2.51 pg/ml) as compared to the control diet (44.50 ± 2.12 pg/ml, P < 0.001, Fig. 4a).VDD significantly decreased testosterone level (0.29 ± 0.26 vs 13.33 ± 1.52 pg/ml, P < 0.001, Fig. 4b) whereas it significantly increased serum homocysteine level (15.66 ± 0.46 vs 9.95 ± 0.91 micromol/L, P = 0.002, Fig. 4c) and serum vitamin B12 level (21,170 ± 3516.1 vs 9168.5 ± 1301.2 pg/ ml, P = 0.02, Fig. 4d).There were no significant differences in serum folate and calcium levels of mice in the VDD group in comparison with the control group (Fig. 4e, f), which confirms that the overfeeding with calcium and phosphorus (see methods) was effective in preventing derangements in calcium and phosphorus homeostasis.www.nature.com/scientificreports/

Effects of vitamin D deficiency on CBS, CSE, and HO-1 expression levels
VDD did not modify the expression of the CBS and CSE genes measured as mRNA (Fig. 5a, c).However, the protein expression of CBS (P = 0.04) and CSE (P = 0.015) was significantly reduced (Fig. 5b, d).Opposite, VDD markedly up-regulated testicular HO-1 both at the mRNA (P = 0.04) and protein (P = 0.011) levels compared to the controls (Fig. 5e, f).Consistent with western blot, the immunofluorescence findings showed low immunoreactivity of CBS and CSE in the testicular germ cells and spermatozoa in VDD-treated mice relative to the controls, while strong immunoreactivity was observed for HO-1 in the VDD group in comparison with the control group (Fig. 5g-i).

Discussion
We tested the effects of vitamin D deprivation on the male reproductive function in a mice model.Our model effectively induced a substantial vitamin D deficiency without causing significant disturbances in calcium metabolism, despite the crucial role of vitamin D in intestinal calcium absorption.It was anticipated that the reduced vitamin D concentration would result in hypocalcemia and subsequent hyperparathyroidism 17 .Therefore, the lack of hypocalcemia observed in our mice with reduced vitamin D levels is intriguing and represents a unique aspect of our model, which closely aligns with the human condition.The prevalence of vitamin D deficiency without accompanying hypocalcemia in humans is noteworthy.It is plausible that any decrease in calcium absorption caused by vitamin D deficiency is counterbalanced by the supplementation of higher quantities of calcium and phosphates, similar to individuals consuming substantial amounts of dairy or other food sources rich in these minerals.Indeed, it was already shown that the calcium deficiency developed by VDD mice can be entirely corrected by calcium supplementation 35 and our results further endorse those findings.The mice under VDD exerted increased body weight, which fits with the already known obesogenic potential of vitamin D deprivation [36][37][38] .This is in contrast with Fu et al. 39 which reported no significant difference in body weight between VDD mice and their controls.Differences in the experimental design, vitamin D-deficient diets, and mouse strains may explain this discrepancy.In any case, our data point to some calcium-independent effects of vitamin D shortage on lipid metabolism.
VDD mice had impaired spermatogenesis associated with signs of oxidative damage and decreased protein levels of the transsulfuration enzymes CBS and CSE whereas both the expression and the protein of the enzyme HO-1 were increased.The impairment of sperm concentration, motility, and morphology from the VDD diet that we recorded is consistent with previous findings in mice [39][40][41] , rats 42 , and humans 16,43 .Shahreza et al. 41   www.nature.com/scientificreports/ a significant decrease in sperm motility after 10 weeks of VDD in mice.Similarly, in VDR-null mice 35 , and in mice with 1α-hydroxylase deletion 44 , resulting in VDD, histological abnormalities of the testis, reduced sperm concentration and motility and enhanced sperm abnormal morphology were observed.This is confirmed by in vitro studies showing that adequate levels of vitamin D are necessary for human sperm 45,46 .Finally, vitamin D regulates cholesterol outflows of human sperm, influences sperm protein serine and threonine phosphorylation, and thus improves sperm survival ability 47 .
The most evident explanation for the impaired spermatogenesis in our study is the deep suppression of testosterone following VDD with vitamin-deficient animals having just 2% of the testosterone circulating in the controls.This is a new and somehow surprising finding.In humans, it was shown that vitamin D supplementation was effective in increasing (already normal) testosterone levels 48 but a following study found no association between vitamin D status and testosterone in young men 49 .However, we showed that in mice the sperm defects were more pronounced in animals with a deeper shortage of vitamin 40 , which was not the case of the above clinical studies, i.e., a deep shortage may be necessary to see effects on testosterone.
Most of the enzymes involved in steroidogenesis have vitamin D response element (VDRE) in their promoters and may indeed suffer low vitamin levels.In a recent study in mice a VDR knock-out model, supposed to abolish the VDR-mediated genomic effects of vitamin D, resulted in a suppression of just 30% of circulating testosterone 50 , which is a lot milder deficit compared to our finding.However, besides genomic effects based on www.nature.com/scientificreports/interaction with the nuclear receptor (VDRn), vitamin D is known to exert non-genomic effects likely dependent on another vitamin D binding molecule in plasma membranes, VDRm 51 .The nuclear and the membrane ligands are different proteins 52 , thus coded by different genes so that the previously reported VDR knock-out model could not block the non-genomic and VDRm-dependent effects.Therefore, VDR knock-out animals still benefit from the non-genomic effects of vitamin D that are instead as well inhibited if the vitamin is not available, which is the case in our model.On this basis, we speculate that the effects of vitamin D on testosterone release are at least in part non-genomic and that these effects occur only in case of deep vitamin deprivation.
Our study on VDD animals revealed notable elevations in sperm ROS, lipid peroxidation, DNA damage, and apoptosis of spermatogenic cells.Prior research has consistently indicated the involvement of vitamin D in apoptosis, with vitamin D deficiency leading to reduced sperm count and motility.These effects are attributed to the diminished proliferation of spermatogenic cells and heightened apoptosis within the same cell population 39,44 .These findings collectively indicate clear evidence of ongoing oxidative damage and disrupted redox metabolism.
OS can impair spermatogenesis with a variety of mechanisms and may explain our findings, however, we need to understand how VDD triggers OS and testosterone decrease.Low testosterone was shown to trigger OS in mice independently of VDD 53 , which fits with our findings, however, a direct effect of VDD on OS was likely in place.The expression of the CBS gene, a master regulator of redox metabolism, is under the control of VDRE so in vitamin D deprivation a lower expression of the gene was expected with decreased expression also of the CSE gene due to lower availability of its substrate.However, in VDD mice we found unchanged levels of the CBS and CSE mRNA whereas it was their protein level to be significantly affected.Thus, post-transcription modifications and/or faster protein catabolism are likely in place, and again, VDD was not acting at the genomic level.On the other side, whatever the mechanism, a shortage of CBS enzymatic activity may explain the reduced testosterone secretion.It has been shown that the CBS product H 2 S regulates testosterone synthesis by inhibiting phosphodiesterase (PDE) expression via sulfhydryl modification and activating cAMP/PKA pathway 54 .
CBS releases H 2 S when working in its alternative modality, which was shown to be activated by CO binding to the heme center of CBS following HO-1 activation by endoplasmic reticulum (ER) stress 27 .Accordingly, in VDD we found increased expression of HO-1 both at the genetic and protein level, which we interpret as the activation of a compensatory, mechanism.VDD has been shown to induce ER stress and vitamin D supplementation to correct it in mice macrophages 55 and in rats' brain 56 , just to mention some evidence and ER stress was likely developing also in our deprived animals.We speculate that such ER stress-activated HO-1 with the aim to trigger corrective actions including H 2 S release.
VDD mice also suffered impaired sperm chromatin maturation with excessive retention of histone proteins and defective protamination.Sperm chromatin maturation is strictly dependent on extensive methylation of DNA and histones 57 , which again points to the one-carbon metabolism: A lack of Hcy re-methylation may cause a deficit of S-adenosyl-methionine and decreased feed to transmethylations paralleled by an accumulation of Hcy.We found indeed increased serum Hcy in our VDD mice.However, we also recorded a huge increase in vitamin B12, which is as well necessary for Hcy re-methylation and is usually decreased in hyperhomocysteinemia.However, the B12 form involved in Hcy re-methylation is methylcobalamin and the activation of B12 to the methylated form is redox dependent, likely mediated by an action of H 2 S released from CBS and CSE 58 .We speculate that B12 accumulated because of reduced conversion to methylcobalamin due to lack of H 2 S, resulting in increased Hcy and deranged methylations leading to sperm chromatin impairment.
A main limitation of the present study is that we did not check our animals for GSH and H 2 S release, therefore we cannot definitely confirm our interpretation.Moreover, our animals suffered very deep vitamin D deprivation leading to extreme testosterone shortage and both these conditions are unlikely to occur in the clinical setting.However, the prevalence of severe clinical vitamin D deficiency is reported to be in double digit frequency and less severe deficits, possibly leading to subtler and undiagnosed clinical consequences, are quite common 60 .Our data further endorse the need for close monitoring, and in case correction, of vitamin D status in infertile men.Moreover, assumed the strong negative effect of the deficiency on testosterone level that we report, the vitamin D status should be included also in the work-up of men with low testosterone level independently of infertility.

Conclusion
In summary, we used a VDD model in mice to demonstrate impaired spermatogenesis due to OS and defective chromatin maturation.A deep reduction in circulating testosterone appeared as a main connection between VDD and sperm defects in our animals.Our data point to the non-genomic and VDRn-independent effects of vitamin D in this context and to a deficit of H 2 S release as a primary effector of the damage in our model, which warrants for further confirmation in duly designed experimental models.Meantime, we call for the need to check the vitamin D status in male infertility and subclinical male hypogonadism.

Animals and study design
This study was approved by the Scientific Ethics Committee of Royan Institute (IR.ACECR.ROYAN.REC.1400.142),and we confirm that all the methods and protocols were carried out according to ARRIVE guidelines and regulations 60 and all methods were performed in accordance with the relevant guidelines and regulations.Sixteen healthy male C57 mice (11-13 g, 4 weeks old) were involved in this study.The mice were kept in the animal house of the Royan Institute for Animal Biotechnology (Isfahan, Iran).All animals were permitted free access to food and water and maintained at 22 ± 2 °C, 45-65% humidity, and a day/night 12 h/12 h photoperiod.After one week acclimatization, mice were randomly divided into two groups (N = 8) as follows: Group 1: (Control group) received a standard chow diet (Standard AIN93G Rodent diet).

Samples collection and assays
At the end of the 14th week, all animals were euthanized by intraperitoneal injection of a cocktail of Ketamine (90 mg/kg body weight) and Xylazine (10 mg/kg body weight) using American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals (https:// www.avma.org) and the final body weight and testicular weight of mice were assessed.Blood samples were acquired using cardiac puncture, transferred to 5 ml vials, and centrifuged at 2500 rpm for 15 min, then the serum portion was separated and frozen at − 20 °C until analysis.The levels of serum testosterone (Testosterone II, Roche, Switzerland), vitamin D3 (Vitamin D total II, Roche, Switzerland), calcium (Calcium C311, CAP, America), folate (Folate III, Roche, Switzerland), and B12 (Vitamin B12 II, Roche, Switzerland) were analyzed using ELISA kits (Elecsys 2010 and Cobas e411 analyzers) according to manufacturer's protocol and absorbance was measured by spectrophotometry at 450 nm.The serum Hcy concentration was measured by high-performance liquid chromatography (HPLC).After separating the epididymis and washing it in PBS at 37 °C, the cauda of the epididymis was incubated in the sperm-washing medium (VitaSperm, Inoclon, Iran) for 30 min at 37 °C to prepare sperm suspension.To decrease observational variances, all parameters were evaluated by a single blinded investigator.

Testicular histopathological assay
The left testis from animals was fixed using the 10% buffered formalin solution overnight.Next, formalin-fixed testes were embedded in paraffin blocks.Paraffinized specimens were cut (5 μm-thick) with a microtome and mounted on glass slides.Next, testicular tissue sections were stained with Hematoxylin and Eosin (H&E) for morphological analysis.Morphological assessment of the testes was carried out with a light microscopic system (CX31 OLYMPUS, Japan) at × 400 magnification 61 .

Sperm analysis
A sperm counting chamber (Makler) was employed for sperm concentration, which was displayed as millions of spermatozoa/ml.Five μl of extracted epididymal spermatozoa were placed in a sperm counting chamber.For each specimen, sperm heads were counted in 5 rows, then counted sperms were divided by 5.For the percentage of sperm motility, 5 μl of the sperm suspensions were located on a pre-warmed slide, and then the motility of 200 spermatozoa was evaluated through visual inspection of the movement types under a light microscope (CX31 OLYMPUS, Japan; × 400 magnification).Finally, the sperm motility percentage was calculated by the formula: Sperm motility (%) = [number of motile sperm (progressive + non-progressive)/total sperm] × 100.Total sperm motility and progressive motility were recorded.

Sperm chromatin maturation assays
For each sperm sample, we evaluated the sperm chromatin maturity using Aniline blue (AB) staining.In brief, 20 μl of washed epididymal spermatozoa were smeared and air-dried at room temperature, then fixed for 2 h in 3% glutaraldehyde in 0.2 M phosphate buffer (pH 7.2).Slides were stained with aqueous AB (5%) in 4% acetic acid (pH 3.5) for 120 min and then washed with PBS.For each slide, 200 sperm cells were randomly counted under the light microscope (× 1000 magnification, CX31 OLYMPUS, Japan), and spermatozoa with unstained nuclei were considered normal (AB−) while those dark blue stained were considered abnormal (AB +) 63 .
Histone replacement by protamine occurs during spermiogenesis.Chromomycin A3 (CMA3) was utilized to assess the sperm protamine levels.For this evaluation, twenty μl of washed epididymal spermatozoa were fixed with methanol: acetic acid (3:1, v/v, Merck, Darmstadt, Germany) for 5 min at − 4 °C.Then, 20 μl of fixed sperm sample was smeared on the slide and allowed to dry at room temperature.Next, smears were stained with 100 µl of 0.25 mg/ml CMA3 solution (Sigma Chemical Co., St Louis, USA) for 1 h.After, each slide was washed with PBS (twice), air-dried, and coated with a coverslip.on each slide, 200 spermatozoa were evaluated by a fluorescent microscope (BX51 OLYMPUS, Japan, × 1000 magnification).Spermatozoa that stained as bright yellow (CMA3 + with deficient protamine) were reported 63 .

Evaluation of sperm DNA damage
Sperm DNA damage was assessed by Acridine orange (AO) staining 64 , which is a fluorescent dye.Briefly, washed spermatozoa were smeared on a slide and air-dried.Then slides were fixed with Carnoy's solution (methanol:acetic acid 3:1, v/v, Merck, Darmstadt, Germany) at 4 °C for 2 h.Next, the slides were washed with PBS and stained with AO stain that was freshly prepared in citrate-phosphate buffer (80 ml 0.1 M citric acid + 5 ml 0.3 M NaH 2 PO 4 , pH 2.5) for 90 min.After, the slides were washed with PBS.At least 200 spermatozoa were evaluated for each sample by a fluorescent microscope (BX51 OLYMPUS, Japan, × 1000 magnification) and DNA of sperms with green fluorescence was assumed as normal double-stranded whereas DNA showing an orange/ red fluorescence was considered as with abnormal denatured DNA.The sperm DNA damage percentage was reported for each sample.www.nature.com/scientificreports/

Apoptosis assessment
Apoptosis was evaluated using a TUNEL kit, following the manufacturer's instructions.Histological sections measuring 5-6 μm were de-paraffinized and rehydrated.Next, the sections were treated with 1 μl of proteinase K (15 μg/ml in 10 mM Tris/HCl, pH: 7.4) for 30 min.The slides were then washed with a Phosphate-buffered saline (PBS) solution.Afterward, the slides were exposed to 25 μl of TUNEL solution, which consisted of a mixture of 50 μl enzyme solution and 450 μl label solution, for 60 min.Following this, the slides were covered with 25 μl of POD-convertor and incubated for 30 min.Subsequently, 25 μl of DAB substrate, composed of 1 μl of DAB and 10 μl of DAB substrate, was added to the slides and incubated for 60 s.Finally, the sections were counterstained with hematoxylin..

Assessment of intracellular ROS production and lipid peroxidation
We assessed sperm membrane lipid peroxidation according to Aitken et al. 67 .In brief, sperm samples (2 × 10 6 cells) were incubated with the final concentration of 5 µM BODIPY C11 for 30 min at 37 °C.Then, samples were washed with PBS (500 g for 5 min), and the sperm lipid peroxidation percentage for each sample was assessed using a FACSCalibur flow cytometer (Becton Dickinson, SanJose, CA, USA).In addition, positive controls were attained after the addition of 2 mM H 2 O 2 to sperm suspensions.

RNA isolation and qRT-PCR protocols
Fifty mg of testicular tissue was homogenized with 1 ml of TRIzol reagent (Yekta Tajhiz Azma, Iran).The RNA concentration and purity were evaluated by reading the absorbance at 230 nm, 260 nm, and 280 nm with a Nan-oDrop1000 spectrophotometer.The cDNA was synthesized from total RNA by cDNA synthesis kit according to the manufacturer's instruction (Biotechrabbit™ cDNA Synthesis Kit).Subsequent Real-time PCR was executed with SYBR green (Yekta Tajhiz Azma, Iran) fluorescent dye using ABI (Applied Biosystems StepOnePlus™).All data were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and expressed as 2 −ΔΔCt68 .The list of primers is revealed in Table 1.

Western blotting
Briefly, protein extraction from the testicular tissue was performed using the TRIzol reagent (Yekta Tajhiz Azma, Iran) 62 .The protein concentration in each sample was estimated by Bradford's assay.For all samples, 30 μg protein was loaded onto 10% sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE) and then transferred to polyvinylidene difluoride (PVDF) membranes.The membranes were blocked in PBS containing 10% skimmed milk powder and incubated overnight, and then rinsed in PBS and probed with the primary antibodies including anti-CBS monoclonal (CBS (B-4): sc-133154, 1:400), anti-CSE monoclonal (CTH (F-1): sc-374249, 1:200), and anti-HO-1 monoclonal (Heme Oxygenase 1(F-4): sc-390991, 1:1000).Afterward, the membranes were washed (three-time, 15 min) in PBS and incubated for 1 h at room temperature with horseradish peroxidase (HRP) conjugated anti-mouse IgG (Dako, P0447, 1:5000) for anti-CBS, anti-CSE, anti-HO-1, and β-actin antibodies.Finally, the presence of the protein bands was identified by an enhanced chemiluminescence system (ECL, Santa Cruz, USA) following the manufacturer's instructions.To reduce costs, membranes were cut into two or three pieces before exposure to the first antibody or the specific antibody.Regarding the repeat, each blot represents at least 4 to 5 samples and they are not repeats of one sample.Data normalization was performed using β-actin as an internal control, and the signal quantification was achieved by Image J software.

Figure 5 .
Figure 5. RT-PCR and western blot of H 2 S-releasing enzymes CBS, CSE, and HO-1 of mouse testis (a-f) in the control and VDD groups.Immunofluorescence of H 2 S-releasing enzymes CBS, CSE, and HO-1 (g-i) of testis sections.The immunofluorescence findings revealed decreased immunoreactivity of CBS and CSE in the testicular germ cells and spermatozoa of VDD-treated mice compared to the control group.Conversely, a notable increase in immunoreactivity for HO-1 was observed in the VDD group when compared to the control group.Data are presented as mean ± SD and analyzed by independent-samples T-test.P < 0.05.CBS cystathionine β-synthase, CSE cystathionine γ-lyase, HO-1 heme oxygenese-1, VDD vitamin D deficiency (N = 5). https://doi.org/10.1038/s41598-023-45986-4 Cystathionine-β-synthase (CBS) GGG CGA AGT GGT CCA TCT C GTT GGC AAA GTC ATC TAC AAGCA 56 Cystathionine-γ-lyase (CSE) GAC TCT ACA TGT CCG AAT GG AAC CTG TAC ACT GAC GCT TCA 56 Heme oxygenase-1 (HO-1) ATG TTG ACT GAC CAC GAC GCC CCA CTT TGT TAG GAA A 56 Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) TGC CGC CTG GAG AAACC TGA AGT CGC AGG AGA CAA CC 60 Intracellular H 2 O 2 level in the caudal epididymal spermatozoa was assessed by Dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay.DCFH-DA is a cell-permeable molecule, that is hydrolyzed to DCFH inside cytosol where it can be oxidized by ROS, to generate 2,7-dichlorodihydrofluorescein (DCF), which can be monitored by fluorescence-based techniques.One million spermatozoa in PBS were incubated with 0.5 μM DCFH-DA at 37 °C for 30 min in a dark condition.Then, sperm H 2 O 2 levels were assessed using a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA).The percentage of sperm cells with intracellular H 2 O 2 was reported for each sample